In the environments of living systems, protein functions are actuated through interaction with other molecules. These interactions, stable or transient, between a protein and another protein or non-protein molecule, enhance or inhibit the stability or function of one, the other, or both partners involved in the interaction. When the interaction results in a stable complex, the complex's association to yet other proteins or non-protein molecules, or a supramolecular assembly, will be affected. When a protein functions as a reagent, its specific interaction with ligands is often exploited to maximize its utility. A case in point is the fortuitous recognition of the mammalian antibody immunoglobulin G (“IgG”) by the bacterial cell wall proteins streptococcal protein G and Staphylococcus aureus protein A.
Genetically encoded sensors that have tightly controlled and subcellularly localized expression have been constructed from polypeptides to exploit the binding characteristics of specific peptide domains (detector) to alter protein fluorescence (reporter) either directly or through resonance energy transfer, requiring a known binding partner of appropriate affinity and specificity. However, in many cases such a detector motif is not known. Examples of this difficulty include the detection of specific phosphoproteins, for which hundreds of variants may exist, and the detection of aberrantly-folded proteins, for which no known binding partners exist.
Green Fluorescent Protein (“GFP”) is a natural fluorescing protein produced by the jellyfish Aequorea victoria. Some amino acid residues in the native protein spontaneously form a fluorophore when the polypeptide is folded into an 11-strand beta-barrel threaded by an alpha-helix running up the axis of the internal cylinder. Because it tolerates N- and C-terminal fusion to a broad variety of proteins, GFP has been used primarily as a fluorescent protein tag, i.e., for making chimeric proteins of GFP linked to other proteins where it functions as an indicator to reveal when, where, and how much of the protein it fuses to is present. In this capacity, it has been expressed in bacteria, yeast, slime mold, plants, Drosophila, zebrafish, and in mammalian cells.
In the jellyfish from which it was isolated, GFP is involved in physiological interactions with the bioluminescent protein aequorin and shifts its blue light absorption to green light emission through energy transfer. In most applications of GFP, this dual-component configuration is not recapitulated, and the excitation of GFP or its derivatives is afforded through optical instrumentation. Other than aequorin, one type of molecule that binds directly to GFP and its derivatives has been developed. These are antibodies, both polyclonal and monoclonal, which are usually used for signal amplification purposes when the GFP signal is too weak, or the protein has been denatured and is no longer fluorescent. In the methods utilizing these antibodies, GFP and its derivatives are treated as generic protein tags, and as such the invention of GFP antibodies and the utility thereof resides within the scope of conventional immuno-chemistry.
The prior art in the GFP related biotechnology has mainly focused on modification of the wild type GFP to increase the intensity of its fluorescence, change the wavelength of its fluorescence, and make its fluorescence conditional. General approaches to achieving these goals include (i) point mutations that change the physical-chemical environment in the vicinity of the fluorophore and (ii) topological rearrangement of the polypeptide chain that results in circular permutated or bipartite versions of the protein. These efforts have yielded many GFP derivatives that, as noninvasive fluorescent markers in living cells, allow for a wide range of applications where it may function as a cell lineage tracer, reporter of gene expression, or as a measure of protein-protein interactions.
It is desirable to find or create ligands that specifically recognize GFP and other fluorescent proteins. It is further desirable that these fluorescent protein-specific ligands can be rationally connected to ligands of other protein or non-protein molecules so that the FP-ligand complex can be recognized by other molecules, indicating the presence or absence of these other molecules.
The present invention is directed to overcoming these and other limitations in fluorescent protein technology.